Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
90
Available online at http://jddtonline.info
RESEARCH ARTICLE
FORMULATION AND EVALUATION OF CANDESARTAN CILEXETIL
TRANSDERMAL PRONIOSOMAL GEL
Dr. A. Seetha Devi, Archana Pinnika*, P. Divya
Department of Pharmaceutics, Hindu college of Pharmacy, Guntur-522002, Andhra Pradesh, India
*Corresponding Author E-mail: [email protected]
ABSTRACT:
The present work deals with the preparation of candesartan cilexetil proniosomal gel by coaservation phase separation
method by using different surfactants, cholesterol and soya lecithin in 9:1:9 and 9:2:9 ratios. The prepared proniosomal gel
formulations were evaluated for vesicle size analysis, surface morphological studies, encapsulation efficiency, In vitro drug
release, ex vivo skin permeation studies and vesicular stability at different storage conditions. The results showed that
candesartan cilexetil in all the formulations was successfully entrapped and a substantial change in release rate and an
alteration in the encapsulation efficiency of candesartan cilexetil from proniosomes were observed upon varying the type of
surfactant and cholesterol content. Vesicles formed with Spans were smaller in size than vesicles formed with Tweens.
Encapsulation efficiency of proniosomes formed from Span 60, Span 40, Span20, and Span 80 was found high compared
with proniosomes prepared from Tweens (Tween 20 and Tween 80). An optimised preparation with 9:2:9 ratio of Span 60,
cholesterol and lecithin gave maximum encapsulation efficiency (92.29%) and showed drug release (95.89±0.26%) in a
controlled manner with a flux value of 1.89 μg/cm2 /hr and permeability co efficient value of 0.094 cm2/hr as compared to
other compositions. No significant changes in relation to vesicle size and encapsulation efficiency were recorded after
stability studies. It is evident from this study that proniosomes are a promising prolonged delivery system for candesartan
cilexetil and have reasonably good stability characteristics.
Keywords: candesartan cilexetil, proniosomes, encapsulation efficiency, flux, stability.
INTRODUCTION:
Transdermal delivery of drugs through the skin to the
systemic circulation provides convenient route of
administration for a variety of drugs. This route is
widely used as it is convenient, safe and avoids the GI
incompatibility, bypassing first pass metabolism,
enhanced bioavailability, decreased frequency of drug
administration. Problem of drug degradation by
digestive enzymes after oral administration, and
discomfort associated with parentral drug administration
can be avoided by this route. For transdermal delivery
of drugs, stratum corneum is the main barrier layer for
permeation of drug moiety and act as a rate limiting
barrier for penetration of drugs. Hence to increase the
permeation of drug through skin membrane, different
approaches of penetration enhancements are used. Drugvehicle based enhancement methods such as pro drugs,
ion-pair, chemical potential of drug, eutectic systems,
complexation and vesicular systems are used in
transdermal research as better alternative method to
enhance permeation of drugs through skin 1.
Vesicular systems such as liposomes and niosomes are
promising systems to enhance the drug delivery through
the skin. They may act as vehicles or as permeation
enhancers for drugs to enhance their penetration via
stratum corneum. Furthermore, they can be used as
controlled percutaneous drug delivery vehicles. The
applicability of these vesicular systems is, however,
limited because of their stability problems. To overcome
the limitations of vesicular drug delivery, proniosomal
approach was introduced, which involved a dry product
or a liquid crystalline gel that could be hydrated
© 2011, JDDT. All Rights Reserved
immediately before use and would avoid many of the
problems associated with niosomal dispersion and
problems of physical stability. Proniosomes are nonionic based surfactant vesicles used to enhance drug
delivery in addition to conventional niosomes. There
are a number of formulation approaches to resolve the
problems of low solubility and low bioavailability.
Proniosome technology offers novel solution for poorly
soluble drugs2.
Candesartan cilexetil (CC) is an esterified prodrug of
candesartan, a non-peptide angiotensin II receptor
antagonist used in the treatment of hypertension. This
medication is also used to treat congestive heart failure.
This drug may also be used to help protect the kidneys
from damage due to diabetes. The major drawback in
the therapeutic application and efficacy of Candesartan
cilexetil as an oral dosage form is its very low aqueous
solubility (0.003 mg/mL) and very low oral
bioavailability i.e. only 15% 3.
Some efforts have been made to enhance the solubility
of candesartan cilexetil to study its effect on the
bioavailability of the drug. A self emulsifying drug
delivery systems (SEDDS) have been developed by M.
Sunitha Reddy et al 4 to enhance the solubility,
diffusion rate and oral bioavailability of candesartan
cilexetil. The solubility of candesartan cilexetil in
different media Tween 80, Sodium dodecyl sulfate,
Macrogol 6000, Sodium carboxymethyl cellulose and
Sodium carboxymethyl dextran has been reported by
Eliska Vaculikova et al 5.Fast dissolving tablets of
ISSN: 2250-1177
CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
Candesartan cilexetil by using Novel co-processed
superdisintegrants have
been developed by S.B.
Shirsand et al6 to increase the solubility and dissolution
rate of drug.
91
Attenuated Total Reflectance Spectroscopy method. The
scanning range was 400-4000 cm-1. The FT-IR spectra
of drug with Non- ionic surfactants,soya lecithin and
cholesterol blends were compared with the FT-IR
spectrum of pure drug.
The aim of this study is to investigate the feasibility of
formulation of proniosomes of Candesartan cilexetil.
Vesicles prepared were characterized by optical,
scanning electron microscopy for vesicle formation and
morphology. Drug encapsulation efficiency and release
studies were carried out. Finally, a stability study of
proniosomal formulations was also performed to
investigate the leaking of the drug during storage.
Solubility studies
The solubility of Candesartan cilexetil was determined
in water and different pH buffers (phosphate buffer of
pH 6.5, pH 6.8 and pH 7.4) and phosphate buffer of pH
6.5 containing different amount of solubilizer (0.3%,
0.35%, 0.4%, 0.5% and 0.7% tween20) and the results
revealed that, solubility of Candesartan cilexetil was
more in phosphate buffer of pH 6.5 containing 0.35%
tween20.
MATERIALS AND METHODS:
Materials
Preparation of Transdermal proniosomal gel
Candesartan cilexetil was obtained as gift sample from
Hetero pharmaceuticals (Hyderabad). Span 60, Span 40,
Span 20 and Span 80 were procured from Loba
chemical pvt. Ltd, Mumbai. Tween 20,tween 80 and
Cholesterol were procured from SD. fine. chemicals
limited, Mumbai. Soya lecithin was procured from Hi
media laboratories PV..Ltd. Mumbai. Absolute ethanol
and Potsium dihydrogen phosphate were procured from
Merck-specialities pvt. Ltd,Mumbai. Sodium hydroxide
was purchased from Thermo fischer scientific india
pvt.Ltd, Mumbai.
Proniosomal gel of candesartan cilexetil was prepared
by coacervation phase separation technique. Drug and
required quantities of materials viz surfactant, soya
lecithin and cholesterol as per the specified ratio were
taken in a dry, clean, wide mouthed test tube. A
measured amount of ethanol (absolute alcohol) was
added to test tube to dissolve the ingredients. The open
end of test tube was covered with a lid to prevent loss of
solvent from it and warmed over water bath at 60-700C
for about 5 minute until the surfactant mixture was
dissolved completely. Then the aqueous phase
(phosphate buffer of pH 6.5 containing 0.35% tween 20)
was added and warmed on a water bath till a clear
solution was formed. The clear solution formed was
cooled to room temperature to convert it to a gel known
as Proniosomal gel. The gel obtained was preserved in
the same glass tube in a dark for characterization
7
.Composition of proniosomal gel formulations were
given in Table 1.
Drug – Excipients Compatability studies by FT-IR
spectroscopy
Fourier-transform infrared (FTIR) spectrophotometer
was used for infra-red analysis of samples to interpret
the interactions of drug with non-ionic surfactants and
other ingredients. The powdered samples were used for
FTIR studies. Infrared (IR) spectra were obtained on a
Bruker 1.2.4 IR system (software OPUS 7.0) using the
Table 1: Composition of proniosomal gel formulations of candesartan cilexetil
Ingredients
FORMULATION CODE
PN
F1
20
PNF
2
20
PNF
3
20
PNF
4
20
PNF
5
20
PNF
6
20
PNF
7
20
PNF
8
20
PNF
9
20
PNF1
0
20
PNF1
1
20
PNF1
2
20
180
_
_
_
_
_
180
_
_
_
_
_
Span 40(mg)
_
180
_
_
_
_
_
180
_
_
_
_
Span 20(mg)
Span 80(mg)
Tween 20(mg)
Tween 80 (mg)
_
_
_
_
_
_
_
_
180
_
_
_
_
180
_
_
_
_
180
_
_
_
_
180
_
_
_
_
_
_
_
_
180
_
_
_
_
180
_
_
_
_
180
_
_
_
_
180
Soya
lecithin(mg)
Cholesterol(mg)
Ethanol(ml)
Phosphate
buffer of pH 6.5
containing
0.35% tween20
(ml)
180
180
180
180
180
180
180
180
180
180
180
180
20
0.4
0.2
20
0.4
0.2
20
0.4
0.2
20
0.4
0.2
20
0.4
0.2
20
0.4
0.2
40
0.4
0.2
40
0.4
0.2
40
0.4
0.2
40
0.4
0.2
40
0.4
0.2
40
0.4
0.2
Candesartan
cilexitil (mg)
Span 60(mg)
© 2011, JDDT. All Rights Reserved
ISSN: 2250-1177
CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
Characterization of Proniosomal gel
Physical appearance of proniosomal gel8
The prepared gel was viewed through naked eye to
characterize color and physical state of gel. Proniosomal
gel was also viewed by optical microscope at 40 X
magnification, to observe crystal characteristics of gel
by spreading as a thin layer on a slide and placing the
cover slip on it. The appearance of each formulation was
checked for its color, consistency and fluidity.
Vesicle Size Analysis9
Size and size distribution studies were done for
niosomes obtained after hydration of proniosomal gel
with agitation (shaking) and without agitation. Size
analysis was done by adding 10ml of phosphate buffer
of pH 6.5 containing 0.35% tween 20
to the
proniosomal gel (100mg) in a small glass vial with
occasional shaking for 10 min. After hydration, the
dispersion of niosomes was observed under optical
microscope at 40x magnification. The sizes of 100
vesicles were measured using a calibrated ocular and
stage micrometer fitted in a optical microscope.
Vesicle morphology10
Shape and surface morphology of proniosomes was
studied using scanning electron microscopy (SEM).
The niosomes formed from the hydration of
proniosomal gel were mounted on an aluminum stub
with double-sided adhesive carbon tape. The vesicles
were then sputter-coated with gold/palladium using a
vacuum evaporator and examined with the scanning
electron microscope equipped with a digital camera at
10kV accelerating voltage.
pH determination11
The proniosomal gel was made into niosomal
suspension. Then the pH of niosomal suspension was
determined using pH meter.
Drug encapsulation efficiency determination12
Proniosomal gel (0.2 g) was reconstituted with 10 ml of
phosphate buffer of pH 6.5 containing 0.35% tween 20
in a glass tube. The aqueous suspension was sonicated
in a bath sonicator for 15 min. The candesartan cilexetil
containing niosomes were separated from unentrapped
drug by centrifugation at 15000 rpm at 20°C for 90 min.
The supernatant was taken and diluted with phosphate
buffer of pH 6.5 containing 0.35% tween20 and the free
drug concentration in the resulting solution was assayed
by UV spectroscopic method at 257nm. The percentage
of drug encapsulation was calculated by using the
following equation.
EP (%) = [(ct - cr)/ct] × 100
Where EP is the encapsulation percentage, Ct is the
concentration of total drug, and Cr is the concentration
of free drug.
In-Vitro drug diffusion study 13
The in-vitro release studies on proniosomal gel were
performed using Franz-diffusion cell. The capacity of
© 2011, JDDT. All Rights Reserved
92
receptor compartment was 10 ml. The area of donor
compartment exposed to receptor compartment was 1.88
cm2. The membrane was mounted between the donor
and receptor compartment. A weighed amount of
proniosomal gel equivalent to 20mg drug was placed on
one side of the membrane. 10 ml of phosphate buffer of
pH 6.5 containing 0.35% tween 20 was taken as receptor
medium. The receptor compartment was surrounded by
a water jacket to maintain the temperature at 37±1ºC.
The receptor fluid was stirred by a Teflon-coated
magnetic bead fitted to a magnetic stirrer at a speed of
600 rpm. Samples were withdrawn at regular intervals (
1, 2, 3, 4,5, 6,7, 8,9, 10, 11, 12, and 24 h). At each
sampling interval, 1 ml samples were withdrawn and
were replaced by equal volumes of fresh receptor fluid.
Sink condition was maintained throughout the
experiment. Samples withdrawn were suitably diluted
and analyzed spectrophotometrically at 257 nm.
Release kinetics14
To study the release kinetics, data obtained from in vitro
diffusion studies was fitted in various kinetic models:
zero order as cumulative percent of drug release vs.
time, first order as log cumulative percentage of drug
remaining vs. time, Higuchi’s model as cumulative
percent of drug release vs. square root of time and
Erosion model as cubic root of the unreleased fraction of
the drug versus time. To determine the mechanism of
drug release, the data was fitted into Korsmeyer and
Peppas equation as log cumulative percentage of drug
released vs. log time, and the exponent n was calculated
from slope of the straight line. The value of n
characterizes the release mechanism of drug, if exponent
is 0.5, then diffusion mechanism is fickian; if 0.5< n
<0.89, mechanism is non- fickian, n = 1 to Case II
(relaxational) transport, and n > 1 to super case II
transport.
Preparation of wistar rat skin for permeation
studies1,11,15
In the present study, wistar albino rat abdominal skin
was used. Ex vivo skin permeation studies were carried
out with Institutional Animal Ethical Committee
approval (Approval Number: HCOP/IAEC/2013-14/01).
After the animal was sacrificed, the abdominal skin was
shaved lightly with an electrical clipper (before of which
a depilatory cream was applied) taking care to prevent
any damage to the surface of the skin, then the
abdominal skin was excised from the animal using a
sharp blade and surgical scissors. The skin was lifted
easily from the animal after incision was made. The skin
was defatted by wiping it with a cotton tip soaked in
diethyl ether to remove the subcutaneous fat and
scraping the dermal side to remove the muscle and
blood vessels. The skin was wiped again with a cotton
tip soaked in ether to prevent any adhering fat. The skin
so prepared was wrapped in aluminium foil and stored
in a deep freezer at (2-5°C) till further use. The skin was
defrosted at room temperature when required.
Ex- Vivo drug permeation studies
The selected formulations on the basis of entrapment
efficiency, and in vitro drug release were subjected to
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Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
permeation studies through rat skin using Franz
Diffusion cell. Before starting the experiment, the skin
was kept in phosphate buffer pH 7.4 for about 1 hour in
a water bath at constant temperature of 37 ºC to allow
water soluble UV absorbing material to leach out. The
effective permeation area of the diffusion cell was 1.88
cm2. 10 ml of phosphate buffer of pH 6.5 containing
0.35% tween 20 was taken as receptor medium. Excised
skin was mounted between the donor and the receptor
compartment with stratum corneum side facing upwards
into the donor compartment. The donor compartment
was left open and wrapped with cellophane to prevent
contamination of the formulation from the atmosphere,
A weighed amount of proniosomal gel equivalent to
20mg drug was placed on the surface of the skin.
Temperature was maintained at 37±1ºC using water
bath. The receptor chamber contents were magnetically
stirred at 600 rpm. Samples were withdrawn at regular
intervals (1, 2, 3, 4,5, 6, 7,8, 9,10, 11,12, and 24 h), and
analyzed for drug content spectrophotometrically at 257
nm. Cumulative amount of drug permeated through the
skin (µg/cm2) was plotted as a function of time (t) for
each formulation.
The skin flux (J)(μg/cm2/h),was determined from the
slope of linear portion of the cumulative amount
permeated per unit area versus the time plot.
Permeability coefficient (Kp) (cm/h), was calculated by
dividing J with the concentration of the drug in donor
cell (C0) by using the following equation:
Kp = J/C0
The skin flux can be experimentally determined from
the following equation J = (dQ/dt) /A
93
2
where J is flux (μg/cm per h), A: Area of skin tissue
(cm2 ) through which drug permeation takes place,
(dQ/dt) is the slope.
Short term stability studies16
Stability studies were conducted for the best formulation
i.e. PNF7 of candesartan cilexetil proniosomal gel. The
ability of vesicles to retain the drug (Drug Retention
Behaviour) was assessed by keeping the proniosomal
gel at three different temperature conditions, i.e.,
refrigeration temperature (4-80C), room temperature
(25±20C) and oven maintained at (45±20C). Throughout
the study, proniosomal formulation was stored in
aluminium foil-sealed glass vials.
The stored
formulation was analyzed for particle size & percent
drug entrapment.
RESULTS AND DISCUSSIONS
Drug – Excipients Compatability studies
The FTIR spectra of pure candesartan cilexetil and
physical mixture of drug with excipient is shown in Fig.
1(a),(b). The presence of peaks at 2932.65 cm-1
(Aromatic C-H stretching), 1115.95 cm-1 (C-N
stretching), 3610.80 cm-1(N-H stretching), 1752.10 cm1
(C=O stretching), 746.98 cm-1(Aromatic C-H bending),
2858.18 cm-1 (Aliphatic C-H stretching), 1241.41 cm-1
(C-O-C) were characteristic to the pure candesartan
cilexetil. IR spectrum of physical mixture of drug with
excipients revealed that there was no appreciable change
in position and intensity of peak with respect to IR
spectrum of pure candesartan cilexetil. IR analysis
revealed that there was no known chemical interaction
between drug and excipients.
Fig 1(a): FT-IR spectrum of pure drug candesartan cilexetil
Fig 1(b): FT-IR spectrum of physical mixture of drug
© 2011, JDDT. All Rights Reserved
ISSN: 2250-1177
CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
Vesicle morphology
The morphology of niosomes derived from proniosomal
gel was studied using Scanning Electron Microcopy.
SEM imaging of candesartan cilexetil revealed that the
niosomes produced from hydration of proniosomes
94
were spherical in shape and discrete with sharp
boundaries having large internal aqueous space. SEM
imaging of niosome produced from
optimized
formulation PNF7 was shown in Fig 2. The optical
microscopy images of the niosomes prepared from
different proniosomal formulations are shown in Fig 3.
(a)
(b)
(c)
(d)
Fig 3 (a),(b),(c),(d): Optical microscopic images of proniosomal gel formulations
Fig 2: SEM images of optimised formulation (PNF7)
Physical appearance of proniosomal gel
Proniosomal gel of candesartan cilexetil prepared with
Span (40, 60) have pale yellow semi solid gel like
appearance. Proniosomal gel produced from Span 20
have thick sticky gel like appearance and span 80 have
light brownish gel like appearance. Proniosomal gel of
tween 20 appears as Yellow highly viscous liquid form
© 2011, JDDT. All Rights Reserved
and proniosomal gel of Tween 80 appears as semisolid
crystalline gel. The results are reported in Table 2.
Vesicle size analysis
Mean Vesicle size of candesartan cilexetil
proniosomes was presented in Table 2, which indicated
that vesicles formed with Spans were smaller in size
than vesicles formed with Tweens. The relationship
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Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
observed between niosome size and Span
hydrophobicity has been attributed to the decrease in
surface energy with increasing hydrophobicity,
resulting in the smaller vesicles. This would also
explain the large vesicle size of niosomes prepared
with Tween which has a much lower hydrophobicity
than Span. Increasing the cholesterol content also
contributed an increase in the hydrophobicity, with a
subsequent slight reduction in vesicle size. The
differences in vesicle size among the niosomes
prepared with Span were not significant. The Size of
vesicle was reduced when dispersion was agitated due
to breakage of larger vesicles to smaller vesicles.
95
As shown in Table 2, Encapsulation efficiency of
proniosomes formed from Span 60, Span 40, Span20,
and Span 80 was found high compared with
proniosomes prepared from Tweens (Tween 20, Tween
80). Among the spans, Span 60 have higher
encapsulation efficiency due to the longer saturated
alkyl chain compared to that of span 40,span 20 and
span 80.Most of the surfactants used to make nonionic
surfactant vesicles such as spans have a low aqueous
solubility. However, freely soluble nonionic surfactants
such as Tween can form the micelles on hydration in
the presence of cholesterol. The tween formulations in
the present study were also able to entrap candesartan
cilexetil efficiently. However the encapsulation
efficiency was relatively low compared to those
composed of Span. This is because the vesicles can be
successfully formed by Tween only in the presence of
excess cholesterol.
pH determination
Skin compatibility is the primary requirement for a
good topical formulation. It was found that the pH of
all the formulations were in the range of 5.64 to 7.20,
which suits the skin pH, indicating skin compatibility.
The results of pH determination are reported in Table
2.
As the cholesterol content of the formulation
increased, the encapsulation of drug also increased.
The formulations (PNF7-PNF12) containing 9:9:2 ratio
of surfactant : soya lecithin : cholesterol showed high
encapsulation efficiency compared to the formulations
(PNF1-PNF6) containing 9:9:1 ratio of surfactant:soya
lecithin :cholesterol.
Drug encapsulation efficiency
Encapsulation efficiency of
proniosomal gel
formulations ranged from 80.40% to 92.20%. The drug
encapsulation efficiency of twelve formulations were
shown in Table 2.and Fig 4.
Table 2: Characterization of proniosomal formulations for physical appearance, pH, Vesicle size and
Encapsulation Efficiency.
Formulation
code
Physical appearance
pH
Mean vesicle size (µm)
Encapsulation
efficiency*
PNF1
Pale yellow semi solid gel
5.89
Without
agitation*
25.06 ± 0.50
With
agitation*
5.97 ± 0.55
85.83 ± 0.67
PNF2
Pale yellow semi solid gel
5.97
24.89 ± 0.40
6.54 ± 0.67
87.65 ±0.47
PNF3
Yellow semisolid thick sticky gel
6.2
19.82 ± 0.16
9.48 ± 0.77
85.79±0.85
PNF4
Light Brownish gel
6.6
17.41 ± 0.94
7.57 ± 0.11
84.08 ± 0.32
PNF5
PNF6
Yellow highly viscous liquid
Yellow semisolid crystalline gel
5.82
7.1
22.50 ± 0.58
20.95 ± 0.96
8.5 ± 0.23
10.64 ± 0.89
82.45 ± 0.45
80.40 ± 0.87
PNF7
Yellowish semisolid gel
6.80
24.65 ± 0.12
4.27 ± 0.43
92.29 ± 0.56
PNF8
PNF9
Yellowish semisolid gel
Yellow semisolid thick sticky gel
6.30
5.64
22.58 ± 0.19
20.62 ± 0.86
5.54 ± 0.38
7.98 ± 0.92
90.21± 0.98
89.56 ± 0.22
PNF10
Light Brownish gel
7.2
18.56 ± 0.54
6.19 ± 0.45
86.24 ± 0.34
PNF11
Brownish semisolid compact mass
6.7
16.22 ± 0.84
7.2 ± 0.67
84.18 ± 0.73
PNF12
Yellow semisolid crystalline gel
6.56
19.45 ± 0.26
9.28 ± 0.87
82.65 ±0.99
*All values represent mean±standard deviations(SD), n=3
© 2011, JDDT. All Rights Reserved
ISSN: 2250-1177
CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
higher surface area due to low vesicle size and hence
better permeability of drug has occured. Higher drug
permeation was seen from niosomes prepared with Span
60 than from the other surfactant. This could be due to
the emulsification effect of the surfactant after the
hydration of the proniosome by the diffusion medium
and formation of channels within the gel structure due
to loss of lipid bilayer. Drug released from proniosomes
prepared with tweens showed lesser extent of drug
release compared to that prepared with span. It might be
due to the larger size of the vesicles and less lipophilic
nature of the Tween, which makes it more difficult for
these vesicles to penetrate or fuse with membrane
whereas the inclusion of Span which is more lipophilic
than Tween further increased the lipophilicity of the
drug leading to better penetration.
% encapsulation efficiency
100
80
60
40
PNF…
PNF…
PNF9
PNF8
PNF7
PNF6
PNF5
PNF4
PNF3
PNF2
PNF1
PNF…
20
0
formulation code
Fig 4: Comparision of entrapment efficiency of
proniosomal gel formulations
Formulations which have higher cholesterol content
(PNF7-PN12) showed less drug release over a period of
24 hrs. Increase in cholesterol ratio resulted in a more
intact lipid bilayers as a barrier for drug release and
decreased its leakage by improving the fluidity of the
bilayers membrane and reducing its permeability, which
led to lower drug elution from the vesicles.
In vitro drug diffusion studies
The results indicated, that PNF1 containing span 60 had
shown higher drug release profile than other
formulations, because niosomes prepared with tweens
and other spans were significantly larger than those
prepared with Span60. Proniosomes of Span 60 were
smaller in size, demonstrated higher hydrophobicity and
% candesartan cilexetil
released
96
100
80
PNF1
PNF2
60
PNF3
40
PNF4
20
PNF5
PNF6
0
0
5
10
15
Time (hr)
20
25
30
Fig. 5: In vitro drug release profiles of candesartan cilexetil from PNF1-PNF6 proniosomal gel formulations
% candesartan cilexetil
released
100
80
PNF7
60
PNF8
PNF9
40
PNF10
20
PNF11
PNF12
0
0
5
10
15
Time (hr)
20
25
30
Fig. 6: In vitro drug release profiles of candesartan cilexetil from PNF7-PNF12 proniosomal gel formulations
© 2011, JDDT. All Rights Reserved
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CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
97
showing that the drug release was controlled mainly by
diffusion mechanism.
In vitro drug release kinetics
In vitro drug release profile of each formulation was
fitted in to different equations and kinetic models to
explain the release kinetics of candesartan cilexetil from
proniosomal gel. When the R2 values of regression plots
for first order and zero order were considered, R2 values
of zero order plots were found to be higher than first
order plots. Hence it is evident that the drug release
from all candesartan cilexetil proniosomal gel
formulations, followed zero order kinetics. By
incorporating release data in Higuchi and Erosion
models, the R2 values of all the formulations were found
to be greater for higuchi model. So all the formulations
in this study were best expressed by higuchi’s classical
diffusion equation. The linearity of plot indicated that
the release process was diffusion controlled. To further
confirm the exact mechanism of drug release, the data
was incorporated in to kores meyer peppas model and
the mechanism of drug release was indicated according
to the value of exponent ‘n’. For all the proniosomal gel
formulations the release exponent ‘n’ value found to be
between 0.5 to 0.89. This indicates the drug released
from all the proniosomal gel formulations followed
non–fickian diffusion. In vitro drug release data was
best fitted in higuchi and kores -meyer peppas equation
Ex- Vivo drug permeation studies
The ex vivo permeation of Candesartan cilexetil through
rat abdominal skin from proniosomal gel formulations
was determined using Franz diffusion cell. Six
proniosomal gel formulations were selected based on
high encapsulation efficiency and in vitro drug release.
The selected proniosomal formulations (PNF1, PNF2,
PNF4, PNF7, PNF8, PNF10) were characterized for
their drug permeation through rat abdominal skin. The
cumulative amount of candesartan cilexetil permeated
per unit area across excised rat skin as the function of
time, flux and permeability
coefficient were
determined.
Increasing the cholesterol content resulted in a more
intact lipid bilayer as a barrier for drug permeation and
decreased its leakage by improving the fluidity of the
bilayer membrane and reducing its permeability, which
led to lower drug elution from the vesicles. Among all
the formulations, PNF1 showed higher drug permeation
81.3±0.76% in 24 hrs with a flux value of 1.90±0.13
µg/cm2 /hr and permeability coefficient of 0.095±0.18
cm2 /hr.
Table 3. Flux and Permeability co efficient of Candesartan cilexetil proniosomal gel formulations
Formulation
code
PNF1
PNF2
Flux (µg/cm2 /hr) *
1.90±0.13
1.87±0.19
Permeability coefficient
(cm2 /hr)*
0.095±0.17
0.088±0.16
PNF4
1.84±0.17
0.087±0.13
PNF7
1.89±0.09
0.094±0.18
PNF8
1.84±0.12
0.092±0.15
PNF10
1.79±0.15
0.089±0.11
* All values represent mean±standard deviations(SD), n=3
Short term Stability studies
Short term Stability studies were conducted for the
optimised formulation (PNF7) of Candesartan cilexetil
proniosomal gel at room temperature (30 ± 2°c), at
refrigerator temperature (4 ± 2°c),and oven maintained
at 45±20c for a period of 2 months. There was no
significant change in relation to vesicle size and
encapsulation efficiency. The results are given in Table
4.
Table 4. Stability study data of optimized formulation (PNF7)
S.No.
Temp.
Initial
After 2 months
Vesicle
size*
Encapsulation
efficiency*
Vesicle
size*
Encapsulation
efficiency*
1
4-80c
4.27±0.43
90.60 ± 0.56
4.37±0.67
89.01±0.46
2
25±20c
4.27±0.43
90.60 ± 0.56
4.98±0.34
86.32±0.56
4.27±0.43
90.60 ± 0.56
5.12±0.95
80.73±0.32
3
0
45±2 c
* All values represent mean±standard deviations(SD), n=3
© 2011, JDDT. All Rights Reserved
ISSN: 2250-1177
CODEN (USA): JDDTAO
Pinnika et al
Journal of Drug Delivery & Therapeutics; 2014, 4(2), 90-98
CONCLUSION:
In the present work, an attempt had been made to
develop proniosomal gel for transdermal delivery of
candesartan cilexetil by using different grades of non
ionic surfactants. All the proniosomal gel formulations
were evaluated for the vesicle size, shape, encapsulation
efficiency, in vitro drug diffusion study, and ex vivo
permeation study and the results were found in the
acceptable range. Vesicle size was decreased with
increasing
concentration
of
cholesterol
and
98
encapsulation of drug was increased with increasing
concentration of cholesterol but drug release was
retarded with increase in concentration of cholesterol.
Among all the formulations PNF7 (span 60, soya
lecithin, and cholesterol present in 9:9:2 ratio) was
selected as an optimised formulation, due to low vesicle
size, high encapsulation efficiency, and released the
drug in a controlled manner for extended period of
time. Overall, these findings indicate that proniosomal
gel will be promising drug delivery system for
candesartan cilexetil.
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ISSN: 2250-1177
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